1. Field of the Invention
The field of this invention is instrumental chemistry as it relates to integrated systems for carrying out combined separation and analytical operations. More specifically, this invention relates to an apparatus and method for integrating a capillary electrophoresis apparatus with an off-column detection system, particularly detection systems using pyro-chemiluminescent techniques.
2. Description of the Prior Art
Capillary electrophoresis (CE) has been highly praised for its capacity to separate sample components using only minute volumes of sample and reagents and yet providing highly resolved separations in extremely short analysis times. The limitation of this technique lies in the relatively few means available with which to conduct highly sensitive analyses on those separated sample components. At present, on-line detection techniques employed with CE can be described based on the location of their analysis as on-column, end-column or off-column.
On-column analytical methods are typically conducted across the capillary either through a window or an opening provided in the capillary wall. On-column techniques such as ultraviolet-visible absorption are in common use because they do not interfere with the separatory operation of the CE and require simple instrumentation. However, as is described in Analyst, "Time-resolved Luminescence Detection of Europium(III) Chelates in Capillary Electrophoresis," Latva, M. et al., Vol. 120, 367-372 (February 1995), the detection limits of the absorption methods are poor due to their dependence on optical path length which is very short across the capillary. The inner diameter of the capillary of a CE is typically in the range of 25 to 100 micrometers.
Alternatively, on-column amperometric detection may provide relatively good detection limits. However, periodic cleaning of the working electrode is required to maintain the detector's performance. This cleaning requirement is accompanied by the additional problem of electronically isolating the working electrode from the high potential field that is applied across the CE capillary during its operation. One solution for isolating the working electrode from the applied potential is to convert the on-column amperometric technique to an end column method. As is described in Analytical Chemistry, "End-Column Detection for Capillary Zone Electrophoresis," Huang, X. and Zare, R. N., Vol. 63, 189-192 (1991) the working electrode may be completely removed from the potential field of the CE by locating a microelectrode adjacent the capillary outlet at the end of the column. However, Huang recognizes that although this arrangement resolves the isolation problem, it is accompanied by losses in both sensitivity and resolution.
Fluorescence is another photometric method for conducting on-column analyses. Conventional fluorescent methods are described in Analytical Chemistry, "Fluorescence Detection in Capillary Electrophoresis: Evaluation of Derivatization Reagents and Techniques," Albin, M. et al., Vol. 63, 417-422 (1991). It is well established that although fluorescent methods can be highly sensitive and selective, the techniques require that the sample components be fluorescent or chemically convertible to a fluorescing compound. These tagging and/or derivatization procedures can be particularly difficult when performed at the low analyte concentrations that are characteristic of CE. Further, these processes can be complex and greatly increase the risk of contaminating the sample. It is also noteworthy with respect to laser induced fluorescence, that the number of fluorescing compounds available for derivatization procedures is further limited by the number of excitation wavelengths of available lasers.
The problem that accompanies any attempt to integrate two or more systems is the interfering effect the systems may have on one another. Background on the mechanism by which CE separates samples into individual components is helpful in understanding the problems that accompany the integration of CE with an off-column detection system.
CE separates the components of a sample on the basis of the electrical properties of those components. A capillary, typically of fused silica, is filled with an ionic buffer solution and the ends of the capillary are emersed in reservoirs containing additional buffer solution. Electrodes are provided at the opposite ends of the capillary and a high voltage potential in the range of 20 to 30 kilovolts is applied. The sample components are caused to migrate under the influence of the applied potential in what is commonly referred to as electroosmotic flow. The unique feature of electroosmotic flow is that it produces a flat profile in the migrating sample across the capillary. Conversely, when a sample is driven through a capillary by hydrostatic pressure, the sample front will have a parabolic profile which increases the occurrence of band broadening and diminishes resolution. The flat profile generated by CE provides enhanced resolution in sample separation over and above that obtainable from chromatographic techniques. Naturally, any convection, currents or pressure differentials existing or created within the CE system will adversely affect the flat profile of the sample as it travels through the capillary. In order to avoid these adverse affects most detection systems employed with CE use either an on-column or end-column approach.
Detectors and methods that require the analyte to be transferred from the capillary outlet to a detector for analysis are referred to as off-column systems. Off-column systems are less known in the art because the connections between the capillary outlet and the detection systems have introduced problems that interfere with the separatory operation of the CE. In addition to the problems discussed above, transfer of CE eluent for off-column detection may include the problems of dead volume and band dispersion due to turbulence and/or resistances to mass transfer. Further still, the transfer is complicated by the fact that the volume of CE eluent will typically be only in the nanoliter range.
Instrumentation Science & Technology, "Fabrication And Evaluation Of Post-Capillary Junctions Via Micro-scale Molding For Use As Reactors And Flow Multiplexers In Capillary Electrokinetic Separations," Staller, T. D. and Sepaniak, M. J., 23(4), 235-254 (1995) describes methods for making and using junctions for integrated systems. The junctions are molded from polymers such as polyimides and epoxys. Specifically, the CE in Staller is integrated with a chemiluminescent/fluorescent technique wherein the chemiluminescent reaction occurs within the molded junction and the light generated by the reaction is transferred by optical fiber to a photomultiplier tube for detection. The problems of dead volume and turbulence due to the addition of reagents are addressed in terms of the geometries of the junction's structure. The junction between the CE and the detection apparatus is described as a closed system, and no means are suggested or disclosed for preventing pressure differentials across the junction or between the CE and detector instrumentation.
Earlier efforts directed at coupling CE to a detector for conducting off-column analysis have been with respect to mass spectrometry, or CE-MS systems. These developments are well documented in Analytical Chemistry, "Capillary Electrophoresis/Mass Spectrometry," Smith, R. D. et al., Vol. 65, No. 13 (Jul. 1, 1993). The CE-MS systems developed have largely relied on an electrospray ionization (ESI) method in which the CE eluent is converted to an electrospray that can be drawn into the mass spectrometer by a high vacuum provided at the mass spectrometer inlet. Recent developments have focused on eliminating the sheathing solution that is used to facilitate the conversion of the CE eluent to a spray. However, because the mass spectrometer operates under an extremely high vacuum, these integrated systems are not capable of controlling the pressure within the interfacing apparatus. Further, these systems generally require a gap between the nebulizer and inlet to the mass spectrometer and thus do not provide a closed interface between the two instruments.
Therefore it is a feature of the present invention to provide an apparatus and method for interfacing a capillary electrophoresis apparatus with an off-column detection system that will not create convection, currents or pressure differentials that would otherwise adversely affect the electroosmotic flow in the capillary of the CE.
It is another feature of the present invention to provide an apparatus and method for interfacing a CE apparatus with an off-column detection system of the type described above in which the pressure within the interfacing apparatus may be adjusted by the introduction and venting of gases of which one may be an inert gas.
It is yet another feature of the present invention to provide an apparatus and method for interfacing a CE apparatus with an off-column detection system of the type described above in which an exit housing is attached to the capillary above the capillary outlet wherein a flow of ionic sheathing buffer is used to complete the electrical circuit of the CE apparatus and to carry the CE eluent into the off-column detector apparatus.
It is still yet another feature of the present invention to provide an apparatus and method for interfacing a CE apparatus with an off-column detection system of the type described above in which the detector is a chemiluminescent detector utilizing pyro-chemiluminescent technique, the furnace of which introduces pressure variations within the interface apparatus which would otherwise interfere with the operation of the CE.
It is still yet another feature of the present invention to provide an apparatus and method for interfacing a capillary electrophoresis apparatus with an off-column detection system of the type described above in which the head pressure at the inlet of the capillary of the CE is equalized with the pressure within the interface apparatus so that a pressure gradient or differential is not created within the CE capillary.
It is still another feature of the present invention to provide an apparatus and method for interfacing a capillary electrophoresis apparatus with an off-column detection system of the type described above in which the head pressure at the capillary inlet of the CE apparatus is controlled so that analyses may be performed at other than atmospheric pressure conditions.